Combined electroporation and microinjection method for the penetration of lipid bilayer membranes

ABSTRACT

Disclosed is a method for penetration of lipid bilayer membranes in order to insert at the tip of a hollow needle-shaped object, such as a micropipet-, into a container formed of a lipid bilayer membrane, wherein said container is placed between said needle-shaped object, with the tip of said needle-shaped object placed in contact with said conainer in such a way that it applies a mechanical force to the lipid membrane of said container, thus mechanically straining it, and a second electrode, whereupon a transient electric pulse of 1-to-10 3  V/cm is applied between the electrodes, resulting in a focused electrical field over said container C which induces a dielectric breakdown of the lipid bilayer causing the needle-shaped object to penetrate the container. Disclosed is also an electroinjection method based on the above method, wherein substances are introduced through the needle-shaped object and into the container after penetration of the needle-shaped object.

This Application claims benefit of international applicationPCT/SE01/02301, filed Oct. 19, 2001 and to Swedish Application No.0003841-4, filed Oct. 20, 2000.

FIELD OF THE INVENTION

The present invention relates to a method for the penetration of lipidbilayer membranes in order to insert tips of needle-shaped objects intolipid membrane enclosed containers, such as cells and liposomes. Theinvention also relates to a method for the injection of a substance intoa lipid bilayer container, such as a cell, or more precisely to amicroinjection method utilizing the concept of electromechanicaldestabilization for the efficient loading of substances, such asbiopolymers, colloidal particles, and other biologically relevantmolecules, into single cell-sized lipid bilayer containers.

BACKGROUND OF THE INVENTION

Today, there is a growing interest in inserting microelectrodes,microcapillaries and micropipet-tip-sensors into single cells. There isalso a growing interest in incorporating sub-micron-sized sensing,sampling, and signal-amplifying particles, as well as large biopolymersinto single cells and liposomes. Several ultrasensitive detection andsensing methods are based directly or indirectly on the use of colloidalparticles. Examples include quantum dot bioconjugate sensors^(1,2), thefamily of Probes Encapsulated By Biologically Localized Embedding(PEBBLE) sensors,³ and silver (Ag) and gold (Au) colloids for use inSurface Enhanced Raman Spectroscopy (SERS) measurements⁴⁻⁶. One of themain limitations for practically using these techniques is thedifficulty of noninvasive and quantitative introduction of colloidalparticles into the cellular interior⁷. Furthermore, it would beattractive to direct the introduction of particles into specificsubcellular compartments such as the cytosol, nucleus, or evenorganelles of individual cells.

GUVs are cell-sized liposomes composed of a single lipid bilayer with anentrapped aqueous compartment⁸. Such liposomes are attractive to use asultra-small reaction containers in which the reaction under study isconfined and separated from the external medium. As such they can beused for studies of biochemical reaction dynamics in compartmentsmimicking a natural intracellular-intraorganellar environment⁹⁻¹².

For use as reaction containers, it is necessary to load vesicles withreactants, including biopolymers like DNA and colloid particles ororganelles (synthetic or naturally derived). Loading of liposomes can,in principle, be performed by adding the particles during thepreparation of the vesicles, since they upon formation trap a part ofthe medium in which they are formed. The trapping efficiency for smallliposomes is, however, limited even for low-molecular-weight compoundsand, entrapment of larger structures such as colloids, is of very lowprobability^(13,14).

Another approach for liposome-loading is to introduce the materials intopreformed vesicles by using micromanipulation-based techniques developedfor loading of single cells. One such technique that is feasible to useis the microinjection technique¹⁵.

By using microneedles made out of pulled glass capillaries with outertip-diameters in the range of 200-500 nm, it is possible to penetratethe membrane wall of a liposome, or cell, and eject controlled volumesof a desired reagent inside the vesicle¹⁶. Injection volumes aretypically in the picoliter to attoliter range and controlled byregulation of injection-time and injection-pressure. The pressure isusually generated by utilization of pressurized-air or oil-hydraulicsystems.

All microinjection techniques are based on mechanical permeabilizationof lipid membranes. When a mechanical point-load is applied, e.g. by acapillary, onto the membrane of a liposome or cell, the membrane isforced to stretch and the isotropic membrane tension, working in theplane of the membrane, is increased. At sufficiently high membranetension, the structural integrity of the liposome, or cell, ismomentarily lost as holes are formed in the membrane, releasing internalfluid in order to counteract the increase in membrane tension. Thismembrane rupture occurs at the site of the highest mechanical load,which is the loci where the point-load is applied, thus allowing theinsertion of a microinjection capillary into the interior of theliposome or cell.

Whereas microinjection works well with certain cell-types andmultilamellar liposomes, there are a few drawbacks to the microinjectiontechniques with unilamellar vesicles and many cell types. Lipid membranebilayers are, typically, very elastic and the absence of internalsupporting structures in unilamellar liposomes make them very difficultto penetrate by mechanical means. The outer diameter of a tip suitablefor injection into thin-walled liposomes and smaller cells is about 200nm, and the inner diameter is typically in the range of only 100nm^(16,17). Such tips are very fragile and extremely difficult to viewin a light microscope, making positioning difficult. The main drawbackof using small inner-diameter injection tips is, however, therequirement of using ultrapure injection liquids in order to preventclogging, limiting injection species to solutions of low- andmedium-molecular-weight compounds. Micro-injection techniques areconsidered to be relatively invasive due to the large mechanical forcesapplied, inducing permanent membrane damage and even lysis of cells andliposomes.

An alternative approach to single-liposome or single-cell loading is touse microelectroporation¹⁸. This technique is based on the theory ofelectro-permeabilization. When exposing a liposome, or cell, to anelectrical field, a potential drop is generated across the membrane. Atsufficiently high field strength, the critical transmembrane potentialV_(c), of the membrane is exceeded, and small pores will form in theliposomal/cellular membrane due to dielectric membrane breakdown. Thetransmembrane potential V_(m), at different loci on the membrane of aspherical vesicle during exposure to a homogeneous electric field ofduration t, can be calculated fromV _(m)=1.5 r _(s) E cos α(1−exp(−t/τ))where E is the electric field strength, r_(s) is the radius of thesphere, α is the angle in relation to the direction of the electricfield, and τ is the capacitive-resistive time constant. Pore formationwill occur at spherical coordinates exposed to the largest potentialshift, which is at the poles facing the electrodes. Typical value forV_(c) for a cell-sized vesicle is ˜1V, and the corresponding electricfield strength needed for exceeding the critical transmembrane potentialV_(c), is in the range of 1-10 kV/cm.

In microelectroporation, the analyte to be encapsulated is added to theexterior solution of the liposomes, or cells, and an electrical field isthen applied locally, using microelectrodes. The amount of analyte thatenters the vesicle is dependent on the analyte concentration gradient,membrane potential, duration of the applied field, and diffusion rate ofthe analyte¹⁹. Drawbacks to the electroporation technique aredifficulties of quantitative loading, and loading of structures of sizeslarger than the effective pore-diameter, which for electropermeabilizederythrocytes is in the range of 1-to-240 nm^(20,21). To improvequantitive loading, controlled amounts of analytes can be introduced viaa small micropipette tip inserted into a hole pre-formed byelectroporation (as described, for example, in JP 8322548). Thisapproach, however, presents a number of disadvantages, including theneed to apply a fairly strong electric field (˜1V) to form a hole fortip insertion.

By combining electroporation and the application of a mechanical forceonto a membrane vesicle, the strength of the applied electrical fieldneeded for membrane permeabilization may be substantially reduced²⁵.This phenomenon is sometimes referred to as electromechanicaldestabilization. It has been shown that electrical fields establishedover lipid bilayer membranes imposes an electrocompressive mechanicalstress σ_(e), acting on the lipid membrane. This force works normal tothe plane of the membrane and leads to a decrease in membrane thickness.If assuming that a lipid membrane behaves as a capacitor, then theelectro-compressive force is proportional to the voltage drop V, overthe membrane and thus to the strength of the applied electric field

$\sigma_{e} = {\frac{1}{2}{{ɛɛ}_{o} \cdot \left( \frac{V}{h_{e}} \right)}}$where ε is the relative dielectric constant and ε₀, is the permitivityand h_(e), is the dielectric thickness of the membrane. The differentialoverall mechanical work dW, done on the lipid membrane is then simplythe sum of the electro-compressive stress σ_(e), and the isotropicmembrane tension T, controlled by the amount of mechanical strainapplied to the membrane

${dW} = {\left\lbrack {\overset{\_}{T} + {\frac{1}{2}{{{ɛɛ}_{o}\left( \frac{V}{h_{e}} \right)} \cdot h}}} \right\rbrack{dA}}$where h is the overall thickness of the lipid bilayer membrane, and dAis the change in membrane area. Consequently, when a mechanical strainis applied to a membrane vesicle, the trans-membrane potential needed toachieve permeabilization can be significantly reduced. Therefore thisapproach for membrane permeabilization may be even less invasive thanelectroporation since lower electric fields can be used, minimizing therisk of unwanted electrochemical reactions at the membrane surface of acell or a liposome.

SUMMARY OF THE INVENTION

The present invention relates to a novel approach for insertingmicropipet tips or any other cylindrical or hollow needle-shaped objectssuch as microelectrodes into containers formed of lipid bilayermembranes, such as cells and liposomes. The basic idea is to destabilizea mechanically strained lipid membrane container with electric pulses,facilitating the penetration of a micropipet.

The invention also relates to a method for introducing substances, suchas large-molecular-weight compounds as well as colloid particles intocontainers formed of lipid bilayer membranes, such as GUVs, cells, andother similar membrane enclosed structures, by applying the concept ofelectromechanical membrane destabilization to a micropipet-assistedmicroinjection technique.

The unique advantage of such an arrangement arises from the combinationof the high degree of spatial- and volume-control of microinjection andthe efficient and non-invasive membrane permeabilization ofelectromechanical destabilization, thereby permitting quantitativeintroduction of analytes, and biologically relevant molecules andparticles including colloids, into unilamellar liposomes and cells.

More specifically, the invention relates to a method for the penetrationof a container formed or surrounded by at least one lipid bilayermembrane in order to insert the tip(s) of at least one hollowneedle-shaped object into said container, wherein said container isplaced between said at least one needle-shaped object, such as anelectrolyte-filled micropipet, equipped with a first electrode, whichpreferably is an internal electrode, and a second electrode, wherein thetip of said at least one needle-shaped object is placed in contact withsaid container in such a way that said tip of said at least oneneedle-shaped object applies a mechanical force to the lipid bilayermembrane of said container, thus mechanically straining said container,whereupon a transient electric pulse of 1-to-10³ V/cm is applied betweensaid first electrode and said second electrode, resulting in a focusedelectric field over said container, said electrical field inducingdielectric breakdown of the lipid bilayer causing the tip of said atleast one needle-shaped object to penetrate the membrane of saidcontainer. Said at least one needle-shaped object is hollow andpreferably constructed from an insulating material and is filled with anelectrically conducting solution or with dispersion of a substance.

Preferably said first electrode is an internal electrode, i.e. locatedinside said hollow needle-shaped object. More preferably, the electrodeis connected to said at least one needle-shaped object, which in thiscase is filled with an electrolyte, in which the electrode is placed.This embodiment has several advantages. For example, no electrochemicalreaction is present at the tip of said at least one needle-shaped objectsince the electrode is located at a distance from the tip of said atleast one needle-shaped object. The presence of such an electrochemicalreaction would otherwise negatively affect the container, especially forcases when the container is a cell since the health and survivability ofthe cell then would deteriorate.

When the tip of the needle-shape object, such as a micropipet tip, haspenetrated the container any of the solution or dispersion contained inthe micropipet may be injected into the container by a variety ofmethods including pressure-induced and electro-osmotic flow, whereuponthe micropipet may be removed, and this is used as a basis for themicroinjection or electroinjection method according to the invention.

The electroinjection technique described here has several distinctadvantages when compared to traditional stab-microinjection protocols.First of all, the technique is less invasive. Since the membrane iselectrically destabilized, less mechanical force is needed forpenetrating the lipid membrane with a micropipet or any otherneedle-shaped object of micro-dimensions. Consequently, there is lessmovement of the injection capillary when located inside a liposome orcell. Such movements may induce severe cell trauma caused by damages tothe cellular matrix.

If compared to electroporation protocols, much lower transmembranepotentials are needed to achieve membrane destabilization, often thegenerated transmembrane potential is only a few mV (which is furtherexplained in the examples below) two to three orders of magnitudesmaller than the V_(m) used in electroporation. Much lower electricfields translates into less electrically induced trauma to the cell, aswell as minimizing the risk of unwanted electrochemical reactions at themembrane surface of a cell or a liposome. The usage of very low electricfield strengths is, beside the fact that the membrane is undermechanical strain, also an effect of the highly focused electric fieldthat is used. When a voltage is applied between the first and the secondelectrode, the non-conducting material of the needle-shaped object, suchas a micropipet, directs the entire established electrical field throughthe tip-end opening of the pipet. Consequently, the part of the lipidmembrane that is in contact with the tip-end of the needle-shaped objectis exposed to the entire electrical field. The coordinates of maximumelectro-compressive force thus spatially coincides with the loci wheremaximum mechanical force is applied.

The presented method for inserting the tips of needle-shape object, suchas micropipets, into containers such as liposomes and cells is much moreefficient than standard stab-microinjection protocols. As a consequence,large diameter micropipets can be used, allowing injections of largestructures into unilamellar vesicles, as well as cells. From this,several possibilities arise. One attractive application is thequantitative introduction of nanosensors¹⁻³ or colloids, for SERSmeasurements⁴⁻⁶ into cells for detection of molecules or for probing ofintracellular structures. Another application is introduction of theseparticles into liposomal reaction containers. Such a procedure wouldallow studies of complex biochemical reactions where the formation ofseveral products and intermediates simultaneously could be monitored. Byincorporation of organelles (naturally or synthetically derived), oreven bacteria into unilamellar vesicles it is possible to create highlyadvanced cell models. This is very attractive for studies of, forexample, complex biochemical signaling systems that translocate betweendifferent intracellular compartments.

The electroinjection technique described here can be performed with veryhigh success rates and allows sequential injection of multiple reagentsinto single liposomes and cells without noticeable leakage. Initiationof complex biochemical reactions inside the confines of a liposome orcell is therefore feasible. This also makes it possible to performultra-small-scale derivatization chemistry inside a liposome, or cell,for analyte labeling prior to microchemical separations.

If combined with the ultra-thin injection needles used for conventionalmicroinjections¹⁷, the method according to the present invention is apowerful technique for introduction of low- and medium-molecular-weightcompounds into smaller cells or even organelles. This is due to theefficient membrane penetrative capacity of the electroinjectiontechnique.

Another application is in the field of so called chip-array injections.Since the technique here presented is highly efficient in terms ofmembrane penetration capacity, it is feasible to construct an arrayinjector system where a plurality of injection needles-tips areclustered together, arrayed, allowing parallel injection of a largenumber of cells simultaneously.

DETAILED DESCRIPTION OF THE INVENTION

As stated above, the present invention relates to a method for thepenetration of lipid bilayer membranes in order to insert at least onetip of at least one needle-shaped object, such as a micropipet tip, intolipid membrane containers. More specifically the method is used for thepenetration of a container constituted of at least one lipid bilayer,wherein said container first is placed between a hollow electrolytefilled, non-conducting needle-shape object, such as a micropipet,equipped with a first electrode, preferably an internal electrode, i.e.an electrode located inside said needle-shaped object, with the tip ofsaid at least one needle-shaped object placed in contact with saidcontainer in such a way that said needle-shaped object applies amechanical force to the lipid membrane of said container, thusmechanically straining said container, and a second electrode,whereupon, by the use of a low-voltage supply, a transient electricpulse of 1-to-10³ V/cm is applied between said first electrode and saidsecond electrode, thus establishing an electric field between said firstand said second electrode, passing through the tip-end of the saidneedle-shaped object, resulting in a highly focused electrical fieldover the membrane part of said container that is in contact with saidneedle-shaped object, said electrical field inducing a local dielectricbreakdown of the part of the mechanically strained lipid bilayer that isin contact with said needle-shaped object, causing the tip of theneedle-shaped object to penetrate the membrane of the container.

Below, the term micropipet is used instead of needle-shaped object,however what is stated for the micropipet is valid also for otherneedle-shaped objects, such as other cylindrical or hollow needle-shapedobjects of appropriate size, such as capillaries, ultra-thin injectionneedles, and microelectrodes. Below, the term also encompasses arrays ofsuch needle-shaped objects, which, for example, may be mounted on achip. Furthermore, the micropipet shall be filled with a conductivemedium to allow electrical contact between the first electrode and thesecond electrode.

Said hollow, non-conducting micropipet, is filled with an electricallyconducting solution or dispersion of a substance. When the micropipethas penetrated the container, any of the solution or dispersion of asubstance contained in the micropipet may be injected into thecontainer, after which the micropipet may be removed. In addition, themicropipet and microelectrode makes a pair of tweezers allowing for themanipulation and subsequent injection into the free-floating cells andliposomes.

Said substance contained in the micropipet and to be injected into acell or liposome may be, for example, a low or medium molecular weightsubstance, such as a dye, a biopolymer, such a DNA, RNA or a protein, acolloidal particle, such as a colloidal bead, a nanosensor, anorganelle, or a bacterium. The expression “low or medium molecularweight substance” relates to a substance with a molecular weight of upto a few kDa, such as up to 3 kDa. The substance is preferably injectedinto the container in the form of a solution or dispersion. A smallvolume, typically 50-to-500×10⁻¹⁵ 1, of the solution or dispersion isinjected into the cell or other unilamellar container.

Said container shall be constituted of or surrounded by at least onelipid bilayer membrane. It may, for example, be a liposome, a vesicle,an organelle, a cell, a multilamellar liposome (MLV) or a giantunilamellar vesicle (GUV). The method according to the invention isparticularly interesting for giant unilamellar vesicles and cells. Thesize of said unilamellar containers shall be organelle or cell-sized,i.e. 0.1-to-10³ μm in diameter.

Said micropipet should be prepared from a nonconductive material inorder to cintain and focus the applied electric field through thetip-end opening (the end closest to the container) of said micropipet,and may, for example, be a glass-, a quartz-, or a plastic-micropipet.The micropipet shall preferably have an outer diameter at the tip of 10nm-to 100 μm, and an inner diameter, i.e. the diameter of the hollowspace inside the micropipet, of 0.05 to 95 μm. Furthermore, themicropipet shall be filled with a conductive medium to allow electricalcontact between the first electrode and the second electrode. Themicropipet shall be equipped with a first electrode, preferably andinternal, highly conductive electrode, such as Pt-, Ag-, Au-, or carbonfiber-electrode, however an electrode of any suitable conductivematerial may be used. The tip of the first electrode, located insidesaid micropipet, should be placed at a distance, preferably 0.5-to-1 cm,from the tip of the micropipet in irder to prevent direct contactbetween said first electrode and the lipid bilayer membrane of thecontainer, thus protecting the membrane from electrochemically generatedreactive species and/or gas bubbles that may form on the first electrodesurface.

Said second electrode may be constituted of any suitable conductivematerial. It may, for example, be a carbon fiber-, a metal-, or aglass-micro-electrode. The second electrode shall preferably have adiameter in the end placed next to the container of approximately1-to-10³ μm. The first and second electrodes may be similar ordifferent. It is also feasible to exchange the second electrode for aground bath-type electrode.

The voltage pulse used to obtain the highly focused electric fieldbetween the first and second electrode, inducing the dielectricbreakdown of the lipid bilayer shall be a transient electric pulse of afield strength 1-to-10³ V/cm. Preferably a transient 0.01-to 10 ms,rectangular waveform dc-voltage pulse of 10-to 60 V/cm is used but otherpulseforms as well as ac-voltage can be used.

Once the micropipet tip is inserted into the lipid membrane container,the micropipet can be used for several different purposes. For example,the micropipet can be used for sampling of intracontainer substances,the micropipet may also be a sensor such as a fiber optic- orelectrochemical-microsensor used for intracontainer measurements or amicroelectrode. Finally the micropipet can be used for injection of asubstance into the container. When a substance is to be introduced intothe container this can be done in many different ways. It is, forexample, possible to use a technique based on electrophoresis,electroendoosmosis, gravity flow, or microinjection with the aid ofcompressed air or oil, or a thermo-sensitive expansion medium.

Once a substance is introduced into the lipid bilayer container, thecontainer can be used for many different purposes. For example, when thesubstance introduced is colloidal particles, such as Ag or Au colloids,quantum dot bioconjugate sensors^(1,2), or PEBBLE sensors³ the containermay be used in conjugation with an ultrasensitive detection or sensingmethod, such as SERS or quantitative fluorescence measurements, fordetection of specific substances.

Another interesting application is the introduction of material intospecific subcellular compartments such as the cytosol, nucleus, or evenorganelles of individual cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Below reference is made to the accompanying drawings on which:

FIG. 1 is a schematic drawing of a capillary holder, consisting of amain body (II) equipped with a Pt wire electrode attached to a connectorpin (III) and an entrance for the microinjector outlet (I). Theinjection tips (VI) are held in place by two rubber O-rings (IV) and ascrewcap (V).

FIG. 2 illustrates electroinjection of flourescein into a giantunilamellar liposome. (A) is a Differential Interference Contrast (DIC)image showing two multilamellar liposomes with two adjacent unilamellarliposomes settled on the coverslip surface. The microelectrode andinjection capillary were positioned in an opposing fashion close to thetarget liposome. (B) illustrates how a mechanical pressure was appliedon the liposome by moving the injection tip towards the microelectrode,forcing the liposome into a kidney-like shape. (C) illustrates how themembrane was permeabilized and the liposome was slid onto the injectiontip and a flourescein solution was injected into the liposome. (D) showshow the injection tip and counter electrode were removed from theliposome. (E) is a fluorescence image of the liposomes after injection.The liposome injected with flourscein is exhibiting strong fluorescencewhile the other liposomes were unaffected. The contour lines of theunilamellar liposomes were digitally enhanced.

FIG. 3 illustrates injection of biopolymer and colloid particles intoGUVs. The figure shows fluorescence images of unilamellar liposomesinjected with highly concentrated solutions of (A) 30 nm fluorescentlatex spheres, (B) small (100 nm) SBL-liposomes (50 μg/ml), stained withDiO, and (C) YOYO-1 labeled T7 DNA (5 ng/ml).

FIG. 4 illustrates electroinjection of YOYO-1-stained T7 phage DNA intoPC-12 cells. (A) and (B) show brightfield and fluorescence images ofcells injected with fluorescent DNA into the cytosol. In (C) and (D) DNAis injected preferentially into the nucleus of the cell and the cytosolonly shows faint fluorescence.

FIG. 5 shows a unilamellar liposome as a reaction container for theintercalation reaction between T2 DNA and YOYO-1. (A) shows aunilamellar protrusion from a multilamellar liposome used as target. (B)shows injection of a solution containing the T2 DNA into the liposome.(C) is a fluorescence image of the DNA-injected vesicle displaying nofluorescence. (D) shows how the injection capillary was withdrawn andreplaced by a thinner capillary loaded with YOYO-1 for a secondinjection. (E) is a fluorescence image after incubation revealing thepresence of fluorescent YOYO-1-intercalated DNA molecules inside theliposome. Brownian motion of micrometer-sized structures could beobserved in the microscope, strongly suggesting that the fluorescenceoriginated mainly from YOYO-intercalated DNA. The YOYO-1 dye, however,also had affinity for the lipid membranes as shown by the strongfluorescence originating from the multilamellar liposome.

EXAMPLES

Materials and Methods

Chemicals

FM 1-43, DiO, YOYO-1 and FluoSpheres (30-nm- and 200-nm-diameter) werefrom Molecular probes. Fluorescein (GC-Grade), T2 DNA (168 000 bp²²), T7DNA (39936 bp²³), L-α-phosphatidylcholine (type II-S), potassiumphosphate (>98%) and Trizma base (>99.9%) were purchased from SIGMA.Chloroform, EDTA (titriplex III), magnesium sulfate and potassiumdihydrogen phosphate (all pro analysi) were obtained from MERCK.Glycerol (>99.5%) from J. T. Baker and deionized water from a Milli-Qsystem (Millipore) was used.

Formation of Small Unilamellar Vesicles (SUVs)

An acetone-purified asolectin preparation dissolved in chloroform wasused²⁴. When preparing SUVs, the lipids were diluted with chloroform toa lipid concentration of 10 mg/ml. For a standard preparation, 300 μl ofthis solution was transferred to a round-bottomed flask. The solvent wasremoved on a rotary evaporator for about 6 h at room temperature. A thincompletely dry lipid film had then formed on the walls of the flask. Tothis film, PBS buffer (Trizma base 5 mM, K₃PO₄ 30 mM, KH₂PO₄ 30 mM,MgSO₄ 1 mM, EDTA 0.5mM, pH 7.8.) containing 1% v/v glycerol, wascarefully added to a lipid concentration of 1 mg/ml. The lipid film wasallowed to swell overnight at 4° C.

Finally, the sample was sonicated in a bath-type sonicator filled withice water.

A total sonication time of about 10 min was normally required before theentire lipid film dissolved and a whitish opalescent mixture was formed.The SUV-suspension was stored at 4° C. and was stable for several days.

Formation of GUVs

The formation of GUVs was performed in a two-step procedure; dehydrationof the lipid dispersion followed by rehydration.

For dehydration, a small volume (5 μl) of SUV-suspension was carefullyplaced on a borosilicate coverslip and placed in a vacuum dessicator at40 C. When the sample was completely dry (no sign of “fluidness” inmicroscope), the dehydration was terminated and the sample was allowedto reach room temperature before rehydration.

The dry sample was first rehydrated with 5 μl buffer. After 3-5 min thesample was further diluted with buffer, this was done very carefully tominimize turbulence in the sample. All rehydration liquids were at roomtemperature.

Micromanipulation and Electroinjection

All injection experiments were performed on an inverted microscope(Leica DM IRB, Wetzlar, Germany) equipped with a Leica PL Fluotar 40×objective and a water hydraulic micromanipulation system (highgraduation manipulator: Narishige MWH-3, Tokyo, coarse manipulator:Narishige MC-35A, Tokyo).

Fluorescence imaging was achieved by sending the output of an Ar⁺-laser(Spectra-Physics 2025-05, 488 nm) through a 488-nm line interferencefilter followed by a spinning disc to break the coherence and scatterthe laser light. The laser light was collected by a lens and was sentthrough a fluorescein filter (Leica 1-3) into the objective to excitethe fluorescent dyes. The fluorescence was collected by the objectiveand detected by a three-chip color CCD camera (Hamamatsu, Kista, Sweden)and recorded on VHS (Panasonic S-VHS AG-5700). Digital image editing wasperformed using an Argus-20 system (Hamamatsu, Kista, Sweden) and AdobePhotoshop graphic software.

The electroinjections were controlled by a microinjection system(Eppendorf Transjector 5246, Hamburg, Germany) and a pulse generator(Digitimer Stimulator DS9A, Welwyn Garden City, U.K.) connected to theinjection capillary.

For translation of liposomes to different locations during theexperiments, carbon fiber microelectrodes (ProCFE, Axon Instruments,Foster City, Calif.) controlled by the micromanipulation system wereused. By simply pushing the vesicles with the microelectrodes, theydetached from the surface and adhered to the electrode tips and could bemoved over long distances to a desired target. With this technique itwas also possible to detach unilamellar protrusion-vesicles that adheredto multilamellar liposomes.

Preparation of Injection Tips

Injection tips were prepared from borosilicate capillaries (length: 10cm, o.d.: 1 mm, i.d.: 0.78 mm; Clark Electromedical Instruments,Reading, UK) that were carefully flame-forged in the back ends in orderto make entrance into the capillary holder easier. The capillaries wereflushed with a stream of nitrogen gas before use. The tips were pulledon a CO₂-laser puller instrument (Model P-2000, Sutter instrument Co.,Novato, Calif.). The outer diameter of the injection tips varied between0.5-2.5 μm. To avoid contamination, tips were pulled immediately beforeuse.

Result and Discussion

Microinjection Procedures

The injection tips were back-filled with a medium of choice and mountedonto an in-house constructed pipet holder shown schematically in FIG. 1.The main purpose of the capillary holder is to secure the injection tipand to act as an interface between the microinjection system and thepulse generator. Basically the device is a standard patch-clamp pipetholder fitted to the outlet of a microinjection system. The main body ofthe pipet holder (II), in this example, was constructed from Plexiglass,and equipped with a Pt-wire electrode connected to a low-voltage pulsegenerator via a connector pin (III). It also comprises an entrance forthe microinjector outlet (I). The injection tips (VI) are firmly held inplace by two rubber O-rings (IV) secured by a Delrin screwcap (V). Thepipet-holder was mounted on the micromanipulation system describedabove. A carbon fiber microelectode with a tip diameter of 5 μm. Afterselecting an appropriate GUY or cell, the injection tip and themicroelectrode were positioned in an opposing fashion, in close contactwith the vesicle at an angle of 10-30° and 150-170° with respect to theobject plane (see FIG. 2). By careful positioning of the electrodes itwas possible to trap free-floating vesicles and subsequently performinjections. By applying a mechanical pressure in terms of moving theinjection tip towards the microelectrode, forcing the vesicle into akidney-like shape (FIG. 2B), it was possible to penetrate the membraneby applying the electric field (a rectangular dc-voltage pulse 40 V/cm,3 ms). When permeabilized, the vesicle slid onto the injection tip andregained its spherical form (FIG. 2C). In this mode, controlled volumesof materials contained in the micropipet could be injected into theliposome. In FIG. 2C, a 25-μm solution of flourscein was injected into asingle liposome. Injection volumes were controlled by the Microinjectionsystem (injection pressure: 250-1000 hPa, time: 0.1-1.5 s). Typically, avolume of 50-to-100 fl was injected into liposomes with a diameter of10-to-20 μm. Injection volumes for cells were kept as small as possiblein order to prevent cell trauma. After completed injection, the tip waswithdrawn from the interior of the vesicle without noticeable signs ofvesicle damage (FIG. 2D) or leakage (FIG. 2E).

GUVs as well as cells were permeabilized in a single-pulse mode, byapplying one or several transient rectangular dc-voltage pulses withpulse durations of 1-10 ms. The electric field strength was typically inthe range of 10-40 V/cm. The membrane voltage V_(m), at different locion the membrane of a vesicle during exposure to a homogeneous electricfield of duration t, can be calculated fromV _(m)=1.5 r _(s) E cos α(1−exp(−t/τ))where E is the electric field strength, r_(s) is the radius of thesphere, α is the angle in relation to the direction of the electricfield, and τ is the capacitive-resistive time constant. Even though thisequation does not exactly match the conditions for the electroinjectiontechnique, it can be used for roughly estimating the transmembranepotential generated. Assuming that a voltage pulse of 40 V/cm is appliedat right angles over a spherical membrane container with a radius of 10μm, a transmembrane potential of only 60 mV is generated. Clearly, sucha small voltage drop across the membrane does not generate sufficientelectro-compressive stress to achieve membrane permeabilization. Sincethe primary electrode is located inside the injection capillary, thecoordinates of electric destabilization spatially coincides with theloci where maximum mechanical force is applied. This electro-mechanicalpermeabilization proved to be a powerful technique for penetration oflipid membranes, allowing the use of coarse micropipet tips.

Results

When using this procedure, we could inject reagents into single cellsand GUVs with diameters of 5-to-25 μm using micropipet tips with anouter diameter of about 2 μm, or up to 3 μm. Therefore, injection intolarger cells can be readily accomplished. Capillaries this coarse alsohave sufficiently large inner diameters for injection of largerstructures and colloid particles at high concentrations into vesicles orcells. This is illustrated by the fact that YOYO-1-labeled T7 phage DNAmolecules (R_(G)=0.56 μm), 30 nm latex spheres as well as100-nm-diameter SUVs were injected into unilamellar vesicles (see FIG.3).

Single PC12 cells were successfully injected with fluorescein (data notshown) as well as T7 phage DNA labeled with YOYO-1 (as shown in FIG. 4).Moreover, preferential but not exclusive injection of materials could beperformed into the cytoplasm (FIG. 4B), and nucleus (FIG. 4D) of singlecells. In both experiments the fluorescence is concentrated locally atthe site of injection, indicating that diffusion of DNA-dye complexthrough the cell was restricted. This shows that the technique herepresented would allow directed delivery of, for example, drugs, geneticmaterial (such as DNA and RNA), proteins, dyes, and particles intospecific compartments of a cell.

Since the electroinjection technique described here can be performedwith very high success rates, it may be a powerful tool for initiationof chemical reactions inside vesicles and cells. This is illustrated bythe experiment shown in FIG. 5. By performing two consecutive injectionsof reagents into a single vesicle, an intercalation reaction between T2phage DNA (R_(G)=1.1 μm) and YOYO-1 was initiated. (A) A unilamellarprotrusion from a multilamellar liposome settled on the coverslip wasselected as target. (B) First a solution containing the T2 DNA (1 ng/ml)was injected into the vesicle using a micropipet tip with an outerdiameter of 2 μm (40 V/cm, 4 ms). (C) Fluorescence image of theDNA-injected vesicle displayed no fluorescence. (D) The injectioncapillary was withdrawn and replaced by a thinner capillary with anouter diameter of 1 μm loaded with YOYO-1 (50 μM), and a secondinjection was performed (20 V/cm, 4 ms). (E) Fluorescence imaging after10 min incubation revealed the presence of fluorescentYOYO-1-intercalated DNA molecules inside the vesicle. Brownian motion ofmicrometer-sized structures could be observed in the microscope,strongly suggesting that the fluorescence originated mainly fromYOYO-intercalated DNA. This experiment illustrates, except from the factthat chemical reactions can be initiated this way, also that it ispossible to sequentially inject multiple reagents into a single vesiclewithout noticeable leakage. Initiation of complex biochemical reactionsinside the confines of a liposome or cell is therefore feasible.

REFERENCES

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1. A method for penetration of lipid bilayer membranes comprising: (i)placing at least one tip of at least one hollow needle-shaped object incontact with a container comprising at least one lipid bilayer membrane,wherein the needle-shaped object comprises a first electrode, andwherein the container is between the needle-shaped object and a secondelectrode; (ii) applying mechanical pressure to the container, wherebythe membrane is mechanically strained but not penetrated; (iii) applyinga transient electric pulse of a field of strength of 1 to 10³ V/cmbetween the first electrode and the second electrode, resulting in afocused electrical field over the container resulting in a dielectricbreakdown of the membrane; and (iv) penetrating the membrane with the atleast one hollow needle-shaped object.
 2. The method according to claim1, wherein said first electrode is located inside said needle-shapedobject.
 3. The method according to claim 1, wherein said needle-shapedobject is a micropipet.
 4. The method according to claim 3, wherein saidmicropipet is a glass micropipet.
 5. The method according to claim 3,wherein said micropipet is a quartz micropipet.
 6. The method accordingto claim 3, wherein said micropipet is a plastic micropipet.
 7. Themethod according to claim 1,wherein said needle-shaped object is amicroelectrode.
 8. The method according to claim 1,wherein saidneedle-shaped object is a capillary.
 9. The method according to claim 1,wherein said container has a diameter of 0.1 to 10³ μm.
 10. The methodaccording to claim 1, wherein the tip of said needle-shaped object hasan outer diameter of 1 nm to 100 μm.
 11. The method according to claim1, wherein the tip of said needle-shaped object has an inner diameterfrom 50 nm to up to 95 mm.
 12. A microinjection method comprisingperforming the method for penetration of lipid bilayer membranesaccording to claim 1, wherein a solution or dispersion of at least onesubstance is delivered through said needle-shaped object and into saidcontainer once the tip of the needle-shaped object has penetrated themembrane.
 13. The method according to claim 12, wherein said substanceis a low or medium molecular weight substance.
 14. The method accordingto claim 12, wherein said substance is a biopolymer.
 15. The methodaccording to claim 12, wherein said substance is a colloidal particle.16. The method according to claim 12, wherein said substance is ananosensor.
 17. The method according to claim 12, wherein said substanceis an organelle.
 18. The method according to claim 12, wherein saidsubstance is a bacterium.
 19. The method according to claim 12, whereinsaid substance is a cell.
 20. The method according to claim 1, whereinsaid membrane is penetrated by an array of several needle-shapedobjects.
 21. The method according to claim 1, wherein said firstelectrode is a Pt-electrode.
 22. The method according to claim 1,wherein said first electrode is an Ag-electrode.
 23. The methodaccording to claim 1, wherein said first electrode is an Au-electrode.24. The method according to claim 1, wherein said first electrode is acarbon fiber-electrode.
 25. The method according to claim 1, wherein thesecond electrode has a diameter of approximately 1 to 10³ μm.
 26. Themethod according to claim 1, wherein said second electrode is a carbonfiber-electrode.
 27. The method according to claim 1, wherein saidsecond electrode is a metal electrode.
 28. The method according to claim1, wherein said second electrode is a glass micro-electrode.
 29. Themethod according to claim 1, wherein said container is a liposome. 30.The method according to claim 1, wherein said container is a vesicle.31. The method according to claim 1, wherein said container is anorganelle.
 32. The method according to claim 1, wherein said containeris a cell.
 33. The method according to claim 1, wherein said containeris a giant unilamellar vesicle (GUV).
 34. The method according to claim1, wherein said container is a multilamellar vesicle (MLV).
 35. Themethod according to claim 12, which is repeated once or several times inorder to insert different substances into saidcontainer.
 36. The methodof claim 1, further comprising delivering a substance to the container.37. The method of claim 36, wherein the substance is delivered bymicroinjection.